Clinical and genetic characteristics predict outcomes of acute myeloid leukemia patients with FLT3 mutations receiving venetoclax‐based therapy

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease, and its heterogeneity is associated with treatment response. Despite the demonstrated success of venetoclax (VEN)‐based therapy for AML, the effect of FLT3 mutations on the efficacy of the therapy is poorly understood. We aimed to compare the efficacy of VEN‐based therapy between FLT3‐mutated (FLT3mut) and FLT3 wild‐type (FLT3wt) patients and identify the predictors of efficacy in FLT3mut patients. Methods A total of 266 AML patients (127 newly diagnosed [ND] and 139 refractory/relapsed [R/R]) receiving VEN‐based regimens were enrolled in this study. A retrospective analysis was performed, and the treatment responses and overall survival (OS) of FLT3mut and FLT3wt patients were compared. Logistic regression and Cox proportional hazards model were applied to examine the clinical and genetic predictors of outcomes. Results With a median of two cycles of VEN‐based therapy, for the ND AML cohort, the FLT3mut group had a comparable composite complete remission (CRc) rate with the FLT3wt group (79.3% vs. 61.2%, p = 0.072). For the R/R AML cohort, the FLT3mut group exhibited a lower CRc rate than the FLT3wt group. With a median follow‐up of 8.6 months (95% confidence interval [CI], 8.0–10), the median OS observed in the FLT3mut and FLT3wt groups for both cohorts were close (14.0 vs. 19.9 months, p = 0.356; 10.0 vs. 11.9 months, p = 0.680). For the ND AML cohort, in FLT3mut patients, MRD‐positive and RNA‐splicing mutation predicted inferior survival (hazard ratio [HR], 10.3; 95% CI: 2.0–53.8; p = 0.006; HR 11.3; 95% CI: 1.2–109.3; p = 0.036, respectively). For the R/R AML cohort, in FLT3mut patients, adverse ELN risk was associated with an inferior response (odds ratio [OR], 0.2; 95% CI: 0.1–0.8; p = 0.025), whereas NPM1 co‐mutation was associated with a superior response (57.1%; OR, 6.7; 95% CI: 1.5–30.1; p = 0.014). CR/CRi predicted a better survival (HR 0.2; 95% CI: 0.1–0.8; p = 0.029), while DNMT3A mutation predicted an inferior survival (HR, 4.6; 95% CI: 1.4–14.9; p = 0.011). Conclusions FLT3 mutations may influence response to VEN‐based therapy in R/R AML patients but not in ND AML patients. Furthermore, clinical and genetic characteristics could predict outcomes of FLT3mut patients receiving VEN‐based therapy.

2][3] Currently, the combination of VEN with hypomethylating agents (HMA) or low-dose cytarabine (LDAC) is recommended as a new standard of care for elderly or unfit AML patients. 46][7][8][9] However, over 30% of ND AML patients and a majority of R/R AML patients still exhibit primary resistance to VEN-based regimens, which may be attributed to the heterogeneity of AML, particularly in terms of genetic variations.1][12] Therefore, exploring the impact of FLT3 mutations on the efficacy of VEN-based regimens is of particular importance.Subgroup analyses conducted in Phase 3 clinical trials have provided insights into the influence of FLT3 mutations on treatment response. 2,13However, there is no detailed comparison between FLT3-mutated (FLT3 mut ) and FLT3 wild-type (FLT3 wt ) patients in the extant literature.Thus, the impact of FLT3 mutations on the efficacy of VEN-based therapy remains uncertain.Previous studies have reported varying composite complete remission (CRc) rates in FLT3 mut patients receiving VEN-based regimens, ranging from 44% to 66.7%.A Phase 3 randomized study demonstrated a CR/CRi rate of 66.7% in 42 FLT3 mut patients, 2,14 and a prospective trial showed a CR/CRi rate of 44.0% in 16 ND FLT3 mut patients receiving VEN plus LDAC. 15In a retrospective study, it was found that the combination of VEN plus HMA treatment resulted in a CR/CRi rate of 60.0% in 50 FLT3 mut cases, including 17 cases of ND and 33 cases of R/R AML 13 .However, it was observed that over 30% of the FLT3 mut patients did not respond to VEN-based regimens, which may be attributed to the clinical and genetic heterogeneity of patients.It is widely recognized that genetics play a significant role in predicting the response to AML treatments. 3,9,16,17Unfortunately, the relationship between genetics and the efficacy of VEN-based therapy in FLT3 mut patients remains unclear.
In this retrospective multicenter study, a total of 266 patients (127 ND AML and 139 R/R AML) who received VEN-based regimens were enrolled to investigate the influence of FLT3 mutations on treatment efficacy.Additionally, we aimed to determine the clinical and genetic predictors of outcomes in FLT3 mut patients.

| Study design and patients
In this retrospective study, we examined consecutive patients diagnosed as ND or R/R AML between July 2018 and April 2022 from 12 hospitals in southern China.These patients received at least one cycle of VEN-based regimens.A total of 277 patients were identified as cases administrated with VEN-based therapy.Eleven patients were excluded due to a lack of genetic data.Finally, 266 patients were enrolled, including 127 ND AML and 139 R/R AML.The treatment efficacy was compared between FLT3 mut and FLT3 wt patients, and co-mutation analysis was conducted only in FLT3 mut patients.The detailed process of screening is depicted in Figure 1.AML was defined based on the World Health Organization's classification. 18Genetic risk was determined according to ELN risk stratification for 2022. 4R/R AML was defined the same as our previous study based on ELN risk stratification for 2017. 9,19Data collection of genetics was conducted before the application of VEN therapy.As we previously reported, 9 the standard metaphase karyotype and fluorescence in situ hybridization analysis were applied to evaluate cytogenetics.A 167-gene panel (Table S1) and a 53-gene PCR panel (Table S2) were used to detect mutations and fusion genes, respectively.Follow-up data consisted of medical records of inpatients and outpatients and telephone records.The institutional review boards approved the ethics of study in accordance with the Declaration of Helsinki.

| Assessment of response
Bone marrow (BM) assessment was done at 28 days after the initiation of VEN therapy.If BM was aplastic, the assessment was conducted again after hematologic recovery.After two cycles of VEN therapy or considered as clinically needed, BM assessments were also done.Disease responses were evaluated as per the 2022 European LeukemiaNet (ELN) criteria for AML and as previously described. 4,9Disease responses include complete remission (CR), CR with incomplete hematological recovery (CRi), morphologic leukemia-free state (MLFS), partial remission (PR), and non-remission (NR).Detailed definitions of responses can be referred to in our previous study. 9Overall response (ORR) comprised CR, CRi, and MLFS, and CRc comprised CR and CRi.Non-remission was defined as a failure to achieve F I G U R E 1 Flow chart.VEN, venetoclax; ND, newly diagnosed; R/R relapsed or refractory.Efficacy comparison was conducted between FLT3 mut and FLT3 wt patients (N = 266), and genetics analysis was only in FLT3 mut patients (N = 67).PR.Once patients gained responses of CR/CRi, measurable residual disease (MRD) of BM aspirate samples was detected by multiparametric flow cytometry.The threshold of MRD was 0.01%.Besides, NPM1, RUNX1-RUNX1T1, and CBFB-MYH11 detected by PCR were also monitored as MRD. 20

| Statistical analysis
The continuous variables of the patients, summarized as range, median, or interquartile range, were analyzed using the Mann-Whitney U test.Categorical variables were summarized as frequencies or percentages and were analyzed using the chi-squared test or Fisher's exact test.
To identify the association of efficacy with clinical and genetic features, logistic regression was performed for treatment response, and Cox proportional hazards model was applied for survival.Overall survival (OS) was defined as the time from initiation of VEN to death, loss of follow-up, and end of follow-up.For survival analysis, Kaplan-Meier curves were applied to different subgroups and compared using log-rank tests.Statistical significance was considered as p < 0.05 for a two-tailed test.All data were analyzed using the EmpowerStats software and the statistical package R (http:// www.r-proje ct.org).Graphs were plotted using GraphPad Prism 8.0.

| Patient characteristics
A total of 277 AML patients were assessed for eligibility, of whom, finally, 266 were enrolled, including 127 ND AML patients and 139 R/R AML patients.FLT3 mutations were detected (ND AML cohort vs. R/R AML cohort) in 29 versus 38 patients, and 98 versus 101 patients were FLT3 wild type, respectively.The study design and mutation landscape of patients are depicted in Figures 1 and 2, respectively.The key baseline and treatment characteristics are summarized in Table 1.For the ND AML cohort, a higher rate of WBC count and a lower rate of adverse cytogenetics were observed in the FLT3 mut group compared with the FLT3 wt group.For the R/R AML cohort, all variables were well-balanced between the two groups.

| Treatment regimens and treatment responses
For induction therapy, 233 patients were treated with VEN + AZA, 20 were treated with VEN + DAC, and 13 were treated with VEN + AZA + HHT.Generally, these regimens consisted of VEN (100 mg on Day 1, 200 mg on Day 2, 400 mg on Days 3-28 for the VEN + AZA group; 100 mg on Day 1, 200 mg on Day 2, 400 mg on Days 3-14 for the VEN + AZA + HHT group), AZA (75 mg/m 2 /day on Days 1-7 subcutaneous) or DAC (20 mg/m 2 /day on Days 1-5 intravenous over 1 h), and HHT (1 mg/m 2 /day on Days 1-7 intravenous over 2 h).The dose of VEN was modified according to the tolerance to therapy and application of therapeutic or prophylactic anti-infective agents. 2 Twentynine patients received sorafenib, including 20 during both induction and posttransplantation and 9 during posttransplantation only.Furthermore, 170 (64.0%) patients experienced sufficient time and dose of VEN during the first cycle.As a result of adverse events, 96 patients had dose interruptions and reductions in the first cycle.Fifty-five patients received subsequent allo-HSCT after induction therapy.Treatment details are presented in Table 1.
In the ND AML cohort, the median OS in FLT3 mut and FLT3 wt groups were close (14.0 vs. 19.9months, p = 0.356, Figure 2A; 14.0 vs. 19.9months, p = 0.790, Figure 2B) regardless of whether the patients who received sorafenib were excluded or not.Patients with FLT3-ITD presented a comparable median OS with patients with FLT3 wt and FLT3-TKD (14.0 vs. 19.9months, p = 0.704, Figure 2C; 14.0 vs. 12.6 months, p = 0.381, Figure 2D).The results of the median OS of the R/R AML cohort were consistent with that of the ND AML cohort.The median OS rates were not significantly different between FLT3 mut and FLT3 wt groups (Figure 2E,F), and the median OS of FLT3-ITD patients was comparable with FLT3 wt or FLT3-TKD patients (Figure 2G,H).

| DISCUSSION
A comprehensive understanding of the influence of FLT3 mutations on the efficacy of VEN-based therapy in patients   As a subset representing poor prognosis, 10,21-23 FLT3 mut patients have been highly concerned these years, especially in the era of targeted therapies.14][15]24 These responses to VEN-based therapy were associated with patients' characteristics, different combined regimens, and patients' genetic features.Regarding the disease setting of patients, Aldoss et al. 13 suggested a CRc rate of 94.0% in ND AML patients and only 42.0% in R/R AML patients.Our results demonstrated that ND AML patients had a significantly higher CRc rate than R/R patients (79.3% vs. 31.6%),which was consistent with Aldoss et al.'s study and our previous study. 9With regard to combined regimens, in ND AML patients receiving VEN + HMA, as per the results of most studies, the CRc rates were from 55.5% to 72.0% [1][2][3]14,24 in FTL3 mut patients. In NDAML patients treated with VEN + LDAC, Wei et al. showed a CRc rate of 44.0% in FLT3 mut patients.Our results were in general agreement with the former, with a CRc rate of 79.3%.Though these studies reported the CRc rates in ND or R/R AML patients with FLT3 mut , only Konopleva et al. 14 compared the CRc rates between FLT3 mut and FLT3 wt ND patients pooled from a randomized Phase 3 study.They found that FLT3 mut patients had comparable CRc rates with FLT3 wt patients (66.7% vs. 66.8%).Our results also suggested that the two groups of ND patients had similar CRc rates, consistent with Konopleva et al.'s findings.However, a new finding in our study is that FLT3 mut patients had higher CRc rates than FLT3 wt patients in the subgroups without IDH1/2 or RUNX1 mutations, which implied that these mutations may impact the treatment response of FLT3 mut patients.We found that FLT3 mut patients exhibited an inferior response compared with FLT3 wt patients in the R/R AML cohort, which indicated that FTL3 mutation had a negative impact on response to VEN-based therapy in these patients.However, this impact was not observed in the aspect of survival.To our knowledge, this is the first real-world study comparing the efficacy between FLT3 mut and FLT3 wt patients, including ND AML and R/R AML individuals.The impact of genetic heterogeneity on response has been documented in patients with ND or R/R AML.3,[5][6][7]9,24,25 Nevertheless, this is still not well explored in the subset of FLT3 mut patients.In the total population, mutations in IDH1/2 were previously reported to be associated with a superior response to VEN-based therapy in both ND and R/R AML patients.For ND AML patients, DiNardo et al. reported CRc rates of 75.4% 2 and 90.9%, 3 and Pollyea et al. reported CRc rates of 79.0% 26 and 90.9% 24 in patients with IDH1/2 mutations.For R/R AML patients, Stahl et al. 5 found that patients with IDH1/2 had a CRc rate of 50.0%, higher than non-IDH1/2 patients.In our previous study, we analyzed 150 patients with R/R AML and noted that IDH1/2 mutations were independent predictors of superior response.9 Regrettably, the association between IDH1/2 mutations and response to VEN-based regimens was not well understood in the subset of FLT3 mut patients.Heretofore, it has only been reported by Konopleva et al., 14 who found that mutations in IDH1/2 were unfavorable factors of response to VEN regimens for FLT3 mut patients, with a CRc rate of 14% (1/7).Contrastingly, our results suggested that co-mutations in IDH1/2 could not predict the treatment response of FLT3 mut patients in both ND AML and R/R AML cohorts.Therefore, further studies with a larger cohort will be needed to confirm and extend the conclusion of this study.NPM1 mutation was a predictor of favorable prognosis in AML.,9 In the subset of FLT3 mut patients, Konopleva et al. 14 showed that patients with co-mutation of NPM1 seemed to have a relatively higher CRc rate than those with the wild type of NPM1, with CRc rates of 70.0% and 58.0, respectively.Aldoss et al. reported 13 a CRc rate of 69.0% (9 out of 13) in NPM1 mut patients, but there was no statistically significant difference from NPM1 wt patients.Our results indicated a CRc of 78.9% in ND NPM1 mut patients, without significant difference from NPM1 wt patients.Thus, the effect of NPM1 mutation on FLT3 mut patients remains controversial in ND AML patients.Interestingly, a new finding in our study is that NPM1 mutation was a favorable predictor of response in R/R AML patients with FLT3 mut .Another new finding in this study is that patients with RUNX1 comutation tended to exhibit an inferior response in the ND AML cohort but did not show any statistical significance, with a CRc of 33.3%.Aldoss et al. reported a CRc of 50.0%, which was higher than ours.The impact of RUNX1 on treatment response in FLT3 mut patients should be considered in further studies with a larger cohort in the future.Our previous study showed that adverse ELN risk was associated with a worse response to VEN regimens for R/R AML.Our results demonstrated that it was also a predictor of response in R/R FLT3 mut patients.However, Aldoss et al. and Konopleva et al. did not analyze the impact of ELN risk on response to VEN therapy.13,14 FLT3 mut AML has been the focus of intense research these years in view of the characteristics of high prevalence, resistance to chemotherapy, easy relapse, and worse survival.21,22,[30][31][32] In the setting of VEN-based regimens, the impact of FLT3 mut on survival has been reported in some studies.Data pooled from Phase 3 trials in ND patients treated with VEN + AZA revealed that the median OS rates were comparable between FLT3 mut and FLT3 wt patients (12.5 months vs. 14.7 months, respectively).14 A retrospective study also showed that FLT3 mut did not change the median OS of R/R AML patients receiving VEN + HMA or LDAC 5 (median OS not reported).Aldoss et al. 13 conducted a retrospective study in 50 patients (17 ND and 33 R/R) with FLT3 mut and found that the median OS was 11.3 months.In this study, the median OS of patients with FLT3 mut was similar to FLT3 wt patients in both ND AML and R/R AML cohorts, which was in line with previous studies.13,14 These might suggest that VEN-based therapy might overcome the poor effect of FLT3 mutation on survival.Co-mutations of FLT3-ITD and DNMT3A had been validated to be associated with worse OS before the emergence of VEN-based therapy.33,34 Our results first reported that DNMT3A mutation was an independent unfavorable factor of survival in FLT3 mut patients receiving VEN-based therapy.This might imply that VEN-based therapy might not overcome the effect of DNMAT3A mutation on the prognosis of FLT3 mut patients.
Our study has a few limitations that should be acknowledged.First, the retrospective nature and possible patient selection bias could not be avoided.Second, the small sample size may have led to bias.Third, the heterogeneity in treatments led to incompletely reliable conclusions.It should be noted that we still observed similar results when patients receiving sorafenib were excluded.To further validate these findings, a prospective and larger sample-size study with longer follow-up is needed in the future.
In conclusion, our findings indicate that FLT3 mutations exert a significant influence on the efficacy of VENbased therapy in patients with R/R AML.Moreover, the presence of IDH1/2 and NPM1 mutations may be indicative of a more favorable response in FLT3 mut patients, while an unfavorable ELN risk profile may be associated with a poorer treatment response.Additionally, the presence of adverse cytogenetics or a mutation in DNMT3A is predictive of worse survival in this patient population.

F I G U R E 2
Survival.(A) OS based on FLT3 mutation in ND patients.(B) OS based on FLT3 mutation in patients without receiving sorafenib in ND patients.(C) OS based on FLT3-ITD and FLT3 wt in ND patients.(D) OS based on FLT3-ITD and -TKD in ND patients.(E) OS based on FLT3 mutation in R/R patients.(F) OS based on FLT3 mutation in patients without receiving sorafenib in R/R patients.(G) OS based on FLT3-ITD and FLT3 wt in R/R patients.(H) OS based on FLT3-ITD and -TKD in R/R patients.The start of follow-up was from the first day of VEN therapy.

T A B L E 2
Outcomes of ND AML patients.

F I G U R E 3
Genetic landscapes and treatment responses in FLT3 mut patients.Patients are grouped by the best response (CR/CRi, MLFS, PR, and NR), annotated with colored bars below the grid.The treatment regimens of each patient are indicated above response bar.The right side of the picture shows the number and composite complete remission (CRc) rate (green bar) of each genetic abnormality.Asterisks indicate genes with p < 0.05 for percent CRc.Active signaling: FLT3-ITD, FLT3-TKD, K/NRAS, PTPN11, and KIT; epigenetic mutations: TET2, DNMT3A, IDH1/2, and TET1; transcription factors: RUNX1, CEBPAmonallelic, GATA2, and CEBPAbiallelic; chromati-cohesin: ASXL1, BCOR, EZH2, and STAG2; RNA-splicing: SF3B1, SRSF2, and U2AF1; and tumor suppressors: TP53, WT1, and PHF6.(A) Genetic landscapes and treatment responses in ND AML patients.(B) Genetic landscapes and treatment responses in R/R AML patients.F I G U R E 4 Multivariate analysis of predictors of survival.*Variables of p < 0.1 in univariate analysis (Tables S6 and S7) were included in this multivariate logistics model.(A) Predictors of survival in ND AML patients.(B) Predictors of survival in R/R AML patients.| 11 of 13 WENG et al.

F I G U R E 5
Survival of FLT3 mut patients.(A) OS based on CR/CRi in ND patients.(B) OS based on CR/CRi in R/R patients.(C) OS based on allo-HSCT in ND patients.(D) OS based on allo-HSCT in R/R patients.(E) OS based on AML type in ND patients.(F) OS based on AML type in R/R patients.(G) OS based on MRD in ND patients.(H) OS based on MRD in R/R patients.(I) OS based on RNA-splicing in ND patients.(J) OS based on DNMT3A in R/R patients.The start of follow-up was from the first day of VEN therapy.
Patient baseline and treatment characteristics.